Cross-reactive influenza a immunization

ABSTRACT

This disclosure relates to methods and compositions for stimulating in an individual an influenza A virus protective response which is subtype cross-protective. Influenza A virus NS1 protein, or a T cell epitope thereof, is administered to the individual in an amount sufficient to stimulate the virus protective response.

GOVERNMENT SUPPORT

The work described herein was funded by PHS Grants ROI-AI19378,ROI-A224750, and T32-AI07272.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 08/419,513filed Apr. 7, 1995 which is is a file wrapper continuation of08/042,884, filed Apr. 5, 1993 now abandoned, which iscontinuation-in-part of Ser. No. 07/564,714, filed Aug. 8, 1990, nowabandoned, the disclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Influenza A virus is a large RNA-containing animal virus. The proteincapsid of the virus is further enclosed in a lipid bilayer-basedenvelope containing protruding spikes of viral glycoprotein. Threeinfluenza A serotypes have been identified (H1, H2 and H3); theclassification based upon differences in the viral glycoprotein.

Upon infection by the Influenza A virus, the body produces antibodies tovariable regions of the surface glycoproteins hemagglutinin (HA) andneuraminidase (NA). This response results in the production ofvirus-specific antibodies which constitute the primary defense of theimmune system. These antibodies provide immunological pressure whichleads to antigenic drift within viral subtypes, as well as shiftsbetween viral subtypes. This relatively high rate of mutagenesis canrender vaccine preparations ineffective because the antigenicdeterminants of the mutated viral proteins can differ significantly fromthose of the protein used as immunogen resulting in the failure of thebody to effectively deal with the infection.

Two central components of the immune system are the B cells and T cells,both of which are lymphocytes. The lymphocyte lineage diverges at theprelymphoblast stage into distinct sublineages. B cells produce andsecrete antibody molecules; a process generally referred to as thehumoral response. T cells are responsible for a variety of cellularresponses referred to generally as cell mediated immune responses.

B cells develop antigen specificity even in the absence of antigenstimulation. It has been estimated, for example, that the preimmunerepertoire of a mouse comprises a class having many millions ofdifferent antibody molecules. This preimmune repertoire is apparentlylarge enough to insure B cell specificity for almost any potentialantigenic determinant.

Current inactivated whole or subunit influenza vaccines provide B cellmediated (humoral) immunity in that they induce antibodies which aredirected toward antigenic determinants of the surface glycoproteins ofthe virus. The first presentation of an influenza antigen to a B cellspecific for the antigen (e.g., at the time of vaccination) results inthe maturation of the B cell into a plasma cell which is highlyspecialized for antibody production. Upon a second encounter with thesame antigen, a rapid and increased secondary response results. Theforeign antigen is bound by the specific antibody followed by clearanceof the bound antigen from the bloodstream.

However, in the case of influenza A, the production ofvirus-neutralizing antibodies provides immunological pressure whichleads to antigenic drift within viral subtypes, as well as shiftsbetween viral subtypes. Vaccines which are directed against antigenicsites do not elicit a broadly cross-reactive (i.e., protective againstall influenza A virus subtypes) B cell response. Furthermore, themutations which result from this immunological pressure can rendercurrent vaccines ineffective.

T cells comprise a class of cells which, although they do not producecirculating antibodies, do play a central role in the immune system. TheT cell class includes helper T cells, cytotoxic T cells and suppressor Tcells. Helper T cells function, in part, by augmenting the response ofother lymphocytes. for example, helper T cells stimulate activated Tlymphocytes, in addition to stimulating B cells activation, by secretinginterleukins as well as other soluble factors. Cytotoxic T cells (alsoreferred to as killer T cells), on the other hand, function bydestroying cells marked with a particular antigen (e.g., cells infectedby virus).

T cells are stimulated by the recognition of a T cell epitope, incombination with a class I or a class II major histocompatibility (MHC)antigen, on the surface of an antigen presenting cell. Macrophagesbelong to the class of antigen presenting cells. Macrophages arephagocytes which ingest foreign particles in the body. These cells arecapable of ingesting even large microorganisms such as protozoa.Following ingestion, the antigen presenting cell digests the foreignparticle and fragments of the foreign particle are displayed on thesurface of the cell.

T cell epitopes differ fundamentally from B cell epitopes. B cellepitopes are antigenic determinants found in the native antigen moleculeand not represented in the denatured antigen or fragments thereof. Tcell epitopes, on the other hand, are found on unfolded molecules orfragments thereof. Furthermore, the T cell epitopes comprise helper Tcell epitopes and cytotoxic T cells epitopes. These epitopes are thoughtto be contained by distinct, albeit possibly overlapping, portions ofthe antigen molecule.

Influenza A virus infection continues to cause epidemics of death andtremendous morbidity throughout the world today even though theetiological agent is known. A great deal of effort has been devoted tothe development of a vaccine, to little avail. A need exists for aneffective influenza A vaccination strategy which could providecross-subtype immunity from Influenza A viral infection.

SUMMARY OF THE INVENTION

This invention relates to Applicants' finding that T cell epitopes ofthe influenza A NS1 protein are capable of stimulating an influenza Avirus protective response, in an individual, which is subtypecross-protective. In a first aspect, the method comprises administeringan effective amount of influenza A virus NS1 protein, in combinationwith a pharmaceutically acceptable carrier thereby stimulating a T cellresponse against an NS1 epitope in the individual resulting in aninfluenza A virus protective response which is subtype cross-protective.A homologue of the NS1 protein in which amino acids have been deleted,inserted or substituted without essentially detracting from theimmunological properties thereof, is also effective for this purpose.

In another aspect, the invention relates to a method and composition forimmunizing an individual against infection by influenza A virus subtypesby the administration of an effective amount of an influenza A virus Tcell epitope in combination with a pharmaceutically acceptable carrier,thereby stimulating a T cell response against the NS1 epitope in theindividual resulting in an influenza A protective response which issubtype cross-protective. The T cell epitope can stimulate a cytotoxic Tcell response, a helper T cell response, or both. A homologue of the NS1T cell epitope in which amino acids have been deleted, inserted orsubstituted without essentially detracting from the immunologicalproperties thereof, is also effective for this purpose.

The invention also relates to an essentially pure oligopeptide having anamino acid sequence corresponding to a T cell epitope of the influenza ANS1 protein. This T cell epitope can stimulate a cytotoxic T cellresponse, and/or a helper T cell response. Again, a homologue of the NS1T cell epitope in which amino acids have been deleted, inserted orsubstituted without essentially detracting from the immunologicalproperties thereof, is also effective for this purpose.

Also disclosed is a method for immunizing an individual againstinfection by influenza A virus subtypes comprising administering aneffective amount of a recombinant virus which expresses the influenza Avirus NS1 protein. The individual can also be immunized by administeringan effective amount of a recombinant virus which expresses an influenzaA virus T cell epitope. These methods are limited to the administrationof a recombinant virus which expresses the NS1 protein or a T cellepitope thereof, thereby stimulating a T cell response against a T cellepitope resulting in an influenza A virus protective response whichsubtype cross-protective.

The methods and compositions described herein provide for a broadlycross-reactive vaccination scheme which is protective against H1, H2 andH3 subtypes of influenza A virus.

DETAILED DESCRIPTION OF THE INVENTION

As discussed previously, influenza A virus comprises three subtypes, H1,H2 and H3. Applicants' invention relates to methods for immunizing anindividual, particularly a human, against infection by any of thesesubtypes. Although the methods described herein are particularly usefulfor human immunization, the methods are equally applicable to othermammals. The term "cross-protective" is used in this application todescribe immunity against H1, H2 and H3 subtypes.

The gene encoding the influenza A NS1 protein has been isolated, cloned,and expressed in a recombinant vaccinia system (see e.g., Bennink etal., J. Virol. 61:1098-1102 (1987)). Using standard biochemicaltechniques (e.g., column chromatography) the NS1 protein, having a knownmolecular weight, can be isolated from cells in which it is expressed.If necessary to attain the desired purity, a hybridoma producingmonoclonal antibody specific for NS1 can be generated. Monoclonalantibody produced by this hybridoma can then be used in an affinitycapture purification scheme.

Homologues of the NS1 protein in which amino acids have been deleted,inserted or substituted without essentially detracting from theimmunological properties thereof can be generated in a variety of ways.For example, in vitro mutagenic techniques can be used to modify thecloned gene encoding the NS1 protein. Such methods, which are well knownto one skilled in the art, can be used to delete, insert or substitutenucleotides in the gene resulting in the deletion, insertion orsubstitution of amino acids in the encoded product. The immunologicalproperties of the mutagenized encoded product can be assayed usingmethods such as those described in the Exemplification which follows.

Effective dosages for the stimulation of an influenza A virus protectiveresponse are determined empirically with initial dosage ranges basedupon historical data for peptide/protein vaccine compositions. As usedherein, the term virus protective refers to an immunological response inthe individual resulting in the successful control or limitation ofinfection by influenza A virus subtypes which is clinically observed.

For example, individuals can be administered dosages of NS1 proteinranging from 0.5-500 micrograms. Whether a particular dosage iseffective can be determined using well known T cell proliferation andcytotoxicity assays. For example, following administration of theprotein to an individual blood is drawn. Cytotoxic T cells areidentifiable by ⁵¹ Cr release assay (see e.g., Kuwano et al., J. Virol.140:1264-1268 (1988)). Helper T cells are identifiable by a standard Tcell proliferation assay (see e.g., Kurane et al., J. Clin. Invest.83:506-513 (1989)). The results from these studies are compared withresults from the same experiments conducted with T cells from the sameindividual prior to administration of the antigen. By comparing thisdata, effective dosage ranges can be determined.

A wide variety of pharmaceutically acceptable carriers are useful.Pharmaceutically acceptable carriers include, for example, water,physiological saline, ethanol polyols (e.g., glycerol or administrationis typically parenteral (i.e., intravenous, intramuscular,intraperitoneal or subcutaneous). An adjuvant (e.g., alum) can also beincluded in the vaccine mixture.

The invention also pertains to a method for immunizing an individualagainst infection by influenza A virus subtypes by administering avaccine composition comprising at least one essentially pure T cellepitope of the NS1 protein in combination with a pharmaceuticallyacceptable carrier. Due to genetic variability between individuals, asingle T cell epitope may not stimulate a virus protective response inall individuals to whom it is administered. Therefore, by combining twoor more distinct T cell epitopes, the vaccine is more broadly effective.As indicated above, helper T cell epitopes and cytotoxic T cell epitopesare thought to comprise distinct (albeit possibly overlapping) regionsof proteins. Cytotoxic T cell epitopes can be distinguished from helperT cells epitopes experimentally using the cytoxicity and proliferationassays described above (helper T cells stimulate proliferation but donot posses cytotoxic activity).

The T cell epitope will be administered as an oligopeptide. Sucholigopeptides can be synthesized chemically following identification ofthe portion of the protein containing the T cell epitope. Alternatively,a truncated portion of the gene encoding the NS1 protein which containsa T cell epitope can be expressed in a cell, and the. encoded productcan be isolated using know methods (e.g., column chromatography, gelelectrophoresis, etc.). In addition, the intact NS1 protein can betreated chemically or enzymatically to generate fragments which containa T cell epitope. Such fragments can be isolated as described above.

As used herein, the term oligopeptide means any amino acid sequencewhich is identical or substantially homologous to a portion of the NS1protein. The expression substantially homologous refers to oligopeptideshaving an amino acid sequence of an NS1 T cell epitope in which aminoacids have been deleted, inserted or substituted without essentiallydetracting from the immunological properties thereof. This definitionincludes amino acid sequences of sufficient length to be classified aspolypeptides (these terms are not used consistently or with greatprecision in the literature).

In a preferred embodiment, both a helper T cell epitope and a cytotoxicT cell epitope are administered to the individual. The stimulation ofcytotoxic T cells is desirable in that these cells will kill cellsinfected by influenza A virus. The stimulation of helper T cells isbeneficial in that they secrete soluble factors which have a stimulatoryeffect on other T cells, as well as B cells. As discussed above, due tothe genetic variability between individuals, it is preferable to includetwo or more cytotoxic T cell epitopes and two or more helper T cellepitopes.

Several methods are described in the literature which are useful for theidentification of T cell epitopes. For example, DeLisi et al. havesuggested that potential epitopic sites may be located by identificationof potential amphipathic alpha helical regions in the molecule. DeLisiet al., Proc. Natl. Acad. Sci. USA 82:7048 (1987). Bixler et al.describe a strategy of synthesizing overlapping synthetic peptidesencompassing an entire protein molecule for delineation of T cellepitopes. Bixler et al., Immunol. Com. 12:593 (1983); Bixler et al. J.Immunogenet. 11:339 (1994). A synthetic method described by Gysen (CibaFoundation Symposium 119:130 (1986)) permits synthesis of a largevariety of peptides thereby mimicking of a variety of potential bindingsites, in turn allowing rapid scanning of a molecule.

More traditional methods, such as enzymatic or chemical digestion ofproteins provide peptide fragments which may be readily tested for Tcell activity. For example, enzymes such as chymotrypsin, elastase,ficin, papain, pepsin, or trypsin provide limited and predictablefragments by cleavage of specified amino acid linkages; similarlychemical compounds such as N-chloro-succinimide BPNS-skatole, cyanogenbromide, formic acid, or hydroxylamine, also produce definable fragmentsby their action on proteins. The presence of the desired T cellstimulating activity in any given fragment can be readily determined bysubjecting purified fragments to a standard T cell proliferation assay,or by analyzing unpurified fragments with a T cell Western Assay. Younget al., Immunol. 59:167 (1986).

In another embodiment, the gene encoding the NS1 protein, or a portionthereof which contains a T cell epitope, can be cloned into arecombinant virus which expresses the NS1 protein, or T cell epitopecontaining portion thereof, in the individual to be immunized. Anexample of such a recombinant virus system is the vaccinia systemdescribed by Paoletti et al. (U.S. Pat. No. 4,603,112), the disclosureof which is incorporated herein by reference. Other viruses have beendescribed in the literature which have a genome which can accommodatethe insertion of a foreign DNA such that a protein encoded by the DNA isexpressed in vivo. Any such recombinant virus is useful for the practiceof this invention.

One skilled in the art will recognize that the compositions describedherein can be combined with the components of influenza A vaccinescurrently in use, thereby resulting in an improved vaccine. Theinvention is illustrated further by the following Exemplification.

EXEMPLIFICATION Example 1

Adoptive Transfer In Mice

Mice

BALB/c mice (H-2^(d)) were purchased from Charles River BreedingLaboratories (Stone Ridge, N.Y.). They were used at 5 to 9 weeks of age.

Influenza Viruses

Influenza A viruses, A/PR/8 (H1N1), A/BZ (H1N1), A/JAP (H2N2), and A/PC(H3N2), or B/HK, were propagated in 10-day-old embryonated chicken eggs.Infected allantoic fluids were harvested 2 days after infection,aliquoted, and stored at -80° (Kuwano, K. et al., J. Immunol.140:1264-1268 (1988)).

Vaccinia Viruses

Vaccinia recombinant viruses containing genes (HA, NP, NS1, and PB2) forA/PR/8 virus were kindly provided by Dr. B. Moss (Bethesda, MD). Theywere constructed and propagated as previously described (Smith, G.L. etal., Virology 160:336-345 (1987)). Briefly, HeLa cells were infectedwith virus for 3 days at 37°. Infected cells were pelleted bycentrifugation, and resuspended in MEM containing 2% FCS. Three cyclesof freezing and thawing were performed and the suspensions were gentlysonicated in water for 1 min followed by trypsinization for 30 min at37°. After centrifugation at 500 rpm for 5 min, supernatants werealiquoted and stored at -80°.

Cells

The cell lines used in this study, P815 cells (H-2^(d) ; mastocytoma)derived from DBA/2 mice, Class 1 MHC molecules, H-2L^(d) - or H-2D^(d)-transfected L929 cell line, and LM1 (K^(k), L^(d), D^(k)) or DM1(K^(k), L^(k), D^(d)), were as described by Weis, J. H. and J. G.Seidman (J. Immunol. 134:1999-2003 (1985)).

Fusion Protein

D proteins were produced in E. coli as described previously (Yamada, A.et al., Escherichia coli. J. Exp. Med. 162:663-674 (1985); Kuwano, K. etal., J. Immunol. 140:1264-1268 (1988)). Briefly, plasmids containing DNAfragments complementary to the viral RNA of A/PR/8 virus weremanipulated to achieve expression of D proteins, which are hybrids ofthe first 81 amino acids of NS1 fused to the 157 amino acids from theC-terminal end of HA2 through a linker of glutamine-isoleucine-proline.After lysis of the bacteria, two 0.1% deoxycholate extractions and one1% Triton X-100 extraction were performed to remove contaminating E.coli proteins, and the D protein was solubilized with 4 M urea at 4° for30 min. The urea was removed by dialysis at 4°. The proteins were storedin 50 mM Tris-HC1, pH 8.0, and 1 mM EDTA. D protein was provided by J.F. Young (Smith, Kline and French Laboratories, Philadelphia, Pa).

CTL Clone

CTL clones were established as described previously (Kuwano, K. et al.,J. Immunol. 140:1264-1268 (1988)). Briefly, CTL responder cells werestimulated weekly with A/PR/8 or D protein-pulsed normal syngeneic.sup.γ -irradiated spleen cells in the presence of 10% Con A stimulatedrat IL2 for several weeks. A limiting dilution was carried out toisolate CTL clones. The B-7 clone was established by stimulation of Dprotein (Kuwano, K. et al., J. Immunol. 140:1264-1268 (1988)). The A-11clone was stimulated by A/PR/8 virus and grew, at a frequency of growth2 out of 96 wells, from a well where two responder cells had beenseeded. For routine passage of clones, 2×10⁶ of clone cell werestimulated weekly by 30×10⁶ of A/PR/8 virus or D protein treated .sup.γ-irradiated spleen cells in the presence of 10% rat IL2 and 5×10⁻⁵ M2-ME.

CTL Assay

P815, LM1, or DM1 cells (2×10⁶) were incubated with 0.5 ml of virus (10⁷-10⁸ PFU) in the presence of ⁵¹ Cr at 37° for 60 min. After threewashings, target cells were incubated for another 1 hr. Then 1×10⁴ 51Cr-labeled target cells were incubated with 1×10⁴ effector cells in atotal volume of 200 μl in 96-well round bottom microplates for 4 hr at37°. The supernatant fluids were harvested and specific lysis wasdetermined as percentage specific lysis=100× (release by CTL-spontaneousrelease)/(maximum release-spontaneous release)!.

Adoptive Transfer of CTL Clone

Cells of the CTL clone (3×10⁶) were suspended in 0.5 ml of RPMI 1640 andinjected into mice via the tail vein. A preliminary experiment indicatedthat transfer of 1.0×10⁶ cells resulted in significant reductions inmean pulmonary virus titers (0.6-0.8 log₁₀ PFU) in recipients of cloneA-11. Six hours after adoptive transfer of the CTL clone, mice wereinfected intranasally with 10³ PFU of virus under ether anesthesia. Thelungs of four mice per group were harvested 3 days later for measurementof virus titers.

Pulmonary Virus Titrations

Virus titrations were performed by plaque formation using MDCK cells aspreviously described (Kuwano, K. et al., J. Immunol. 140:1264-1268(1988)). Briefly, infected lungs taken from recipient mice were manuallyhomogenized in 1.5 ml of PBS containing 0.1% BSA. After centrifugation,the lung supernatants were serially 10-fold diluted in PBS. Dilutedvirus samples (100 μl ) were added to confluent MDCK cells in 24-welltissue culture plates and incubated at 37° for 1 hr. Each well thenreceived 1 ml of 1% agar prepared as described earlier (Kuwano, K. etal., J. Immunol. 140:1264-1268 (1988)). After 2 days of incubation, 1 mlof 10% neutral red (GIBCO, Chagrin Falls, Ohio) in PBS was overlaid onthe agar in the wells. Plaques were counted 8 hr later. The results wereexpressed as the mean log₁₀ PFU/ml of duplicate samples.

Cross-Reactivity of Clone A-11 Stimulated by A/PR/8 Virus

Four CTL clones were established that were derived from A/PR/8virus-immune spleen cells of BALB/c mice (H-2^(d)) stimulated by A/PR/8virus (H1N1). Two of the CTL clones demonstrated H1 subtype-specificlysis of virus-infected target cells. These CTL clones were PB2 proteinspecific as determined using target cells infected with a vacciniarecombinant virus containing the gene for PB2 of A/PR/8 virus. Clone1E8, representative of two subtype H1-specific clones, is shown as anegative control in Table 2. Clone A-11, which is representative of theother two CTL clones, demonstrated cross-reactive lysis of target cellswhich were infected with A/PR/8 (H1N1), A/BZ (H1N1), A/JAP (H2N2), orA/PC (H3N2) viruses, but failed to lyse B/HK-infected target cells(Table 1). The B-7 CTL clone (Kuwano, K. et al., J. Immunol.140:1264-1268 (1988)) was used as a control. B-7 had been stimulated bya fusion protein containing part of the HA2 subunit of A/PR/8 virus andshowed subtype H1H2 cross-reactive lysis of target cells that had beeninfected with A/PR/8 (H1), A/BZ (H1), or A/JAP (H2) viruses. Thephenotypes of the cell surface antigens of both the A-11 and B-7 cloneswere Thyl+, Lyt-2+, and L3T4.

                  TABLE 1                                                         ______________________________________                                        Virus Specificity of Clone A-11 Stimulated by                                 A/PR/8 Virus                                                                          % Specific Lysis of P815 Target Cells                                               A/PR/8  A/BZ  A/JAP A/PC       Unin-                            Clone                                                                              E/T Ratio                                                                              (H1N1)  (H1N1)                                                                              (H2N2)                                                                              (H3N2)                                                                              B/HK fected                           ______________________________________                                        A-11.sup.a                                                                         1.0      54      59    58    62    0    0                                     0.5      43      51    43    43    0    0                                B-7.sup.b                                                                          1.0      62      65    43    0     1    0                                     0.5      45      52    32    0     0    0                                ______________________________________                                         .sup.a Clone A11 expresses 94% of Thy1.2, 86% of Lyt2, and 5% of L3T4         surface Ag.                                                                   .sup.b Clone Bp7 expresses 97% of Thy1.2, 95% of Lyt2, and 0% of L3T4         surface Ag.                                                              

CTL Clone A-11 is NS1-Protein Specific

To examine the influenza protein recognized by clone A-11, target cellswere infected with recombinant vaccinia viruses containing variousinfluenza genes of A/PR/8 virus and were used in CTL assays. As shown inTable 2, clone A-11 significantly lysed NS1-VAC-infected and A/PR/8virus-infected P815 target cells as positive control. However, cloneA-11 failed to recognize HA-VAC, NP-VAC, PB2-VAC, or parentalVAC-infected P815 target cells. Clone B-7 as a negative control lysedHA-VAC-infected target cells as well as A/PR/8 virus-infected targetcells, but did not lyse NP-VAC-or VAC-infected target cells. Clone 1ES,also derived from A/PR/8 virus-immune spleen cells by repeatedstimulation with A/PR/8 virus as described above, recognizedPB2-VAC-infected target cells or A/PR/8 virus-infected target cells, butfailed to recognize NS1-VAC or HA-VAC-infected target cells; it is alsoincluded as a control. These results indicated that CTL clone A-11recognizes the NS1 protein on influenza A virus-infected cells.

                  TABLE 2                                                         ______________________________________                                        Recognition of NS1 Protein of A/PR/8                                          Virus by Clone A-11                                                           % Specific Lysis of P815 Target Cells                                              E/T            HA-         NS1- PB2-      Unin-                          Clone                                                                              Ratio  A/PR/8  VAC  NP-VAC VAC  VAC  VAC  fected                         ______________________________________                                        Experiment 1                                                                  A-11 1.0    55      -4   ND     42   -4   ND   -1                                  0.5    47      -4   ND     33   -2        -2                             1E8  1.0    61      -3   ND      0   62   ND   -1                                  0.5    53      -4   ND      0   47   ND   -1                             Experiment 2                                                                  A-11 3.0    78       0   -1     ND   ND    0    1                                  1.0    75       1   -1     ND   ND    0    1                             B-7  3.0    91      91   -1     ND   ND   -2    0                                  1.0    72      80   -1     ND   ND   -1    0                             ______________________________________                                    

Reduction of Pulmonary Virus Titers by Transfer of NS1-Specific CTLClone

To examine whether adoptive transfer of NS1 protein-specific CTL cloneA-11 would reduce virus titers in the lungs of mice infected withinfluenza viruses, 3×10⁶ cells of clone A-11 were adoptively transferredto BALB/c mice 6 hr prior to influenza infection. Three days later,lungs were removed for titration of influenza viruses. Virus titrationswere performed by plaque formation assays in MDCK cells. Similar resultswere obtained in two experiments with mean decreases in pulmonary virustiters of about 1.0 log₁₀. As shown in Table 3, adoptive transfer of CTLclone A-11 significantly reduced the virus titers in the lungs of miceinfected with A/PR/8, A/JAP, or A/PC viruses, but did not reduce thevirus titer in the lungs of mice infected with B/HK virus. These resultsreflect the in vitro cross-reactivity of CTL clone A-11 shown in Table1.

                  TABLE 3                                                         ______________________________________                                        Reduction of Pulmonary Virus Titers by                                        Adoptive Transfers of Clone A-11                                                                      RECIPIENTS                                            CTL        Virus       Virus Titer in Lungs.sup.b                             Clone A-11.sup.a                                                                         Challenge   Experiment 1                                                                             Experiment 2                                ______________________________________                                        +          A/PR/8 (N1N1)                                                                              5.1 ± 0.4.sup.c                                                                      .sup. 5.7 ± 0.2.sup.f                    -          A/PR/8      6.2 ± 0.3                                                                             6.8 ± 0.2                                +          A/JAP (H2N2)                                                                              .sup. 3.0 ± 0.6.sup.d                                                                 .sup. 3.3 ± 0.2.sup.g                    -          A/JAP       4.3 ± 0.2                                                                             4.4 ± 0.2                                +          A/PC (H3N2)  4.0 ± 0.4.sup.e                                                                      .sup. 4.5 ± 0.4.sup.h                    -          A/PC        5.0 ± 0.1                                                                             5.7 ± 0.2                                +          B/HK        4.1 ± 0.1                                                                             ND                                          -          B/HK        4.2 ± 0.1                                           ______________________________________                                         .sup.a Cells (3 × 10.sup.6) were transferred 6 hr before virus          challenge; +, transferred; -, no cells transferred.                           .sup.b Lungs were taken and virus titers were examined by plaque assays i     MDCK cells 3 days after virus challenge.                                      .sup.c P < 0.01, Student's t test.                                            .sup.d P < 0.02, Student's t test.                                            .sup.e P < 0.005, Student's t test.                                           .sup.f P < 0.005, Student's t test.                                           .sup.g P < 0.0005, Student's t test.                                          .sup.h P < 0.005, Student's t test.                                      

MHC Restriction of Target Cell Lysis by Clone A-11

L929 cells (H-2^(k)) transfected with genes encoding H-2D^(d) (DM1cells) and H-2L^(d) (LM1 cells) were used to examine the MHC restrictionof target cells lysis by CTL clone A-11. As shown in Table 4, CTL cloneA-11 significantly lysed A/PR/8 virus-infected LM1 (H-2L^(d)) targetcells, but failed to lyse A/PR/8 virus-infected DM1 (H-2^(d)) or A/PR/8virus-infected DAP (H-2^(k)) target cells.

As a control, CTL derived from bulk cultures of A/PR/8 virus-immuneBALB/c (H-2d) spleen cells that had been stimulated by A/PR/8 virus inthe presence of IL2 for several weeks were also used in this experiment.These virus-stimulated CTL lysed LM1 or DM1 target cells infected withA/PR/8 virus, but did not kill A/PR/8 virus-infected DAP target cells.It was also observed that the CTL clone A-11 was unable to recognizeA/PR/8 virus-infected peritoneal exudate cells of C3H.OL mice (H-2K^(d),D^(k)). These results indicate that recognition by the CTL clone A-11 ofNS1 on A/PR/8 virus-infected target cells is restricted by the H-2L^(d)allele.

                                      TABLE 4                                     __________________________________________________________________________    MHC Restriction of CTL Recognition by CTL Clone A-11                                    % Specific Lysis of Target Cells                                                        DM1 (H-                                                             LM1 (H-2K.sup.k, D.sup.k, L.sup.d)                                                      2K.sup.k, D.sup.k, D.sup.d)                                                             DAP (H-2K.sup.k, D.sup.k)                       CTL  E/T Ratio                                                                          A/PR/8                                                                             None A/PR/B                                                                              None                                                                              A/PR/8                                                                             None                                       __________________________________________________________________________    A-11 5.0  58   1    9     2   0    0                                               2.5  43   1    2     2   2    0                                          A/PR/8                                                                             5.0  36   2    24    2   1    1                                          stimu-                                                                             2.5  30   1    11    1   1    1                                          lated                                                                         CTL                                                                           __________________________________________________________________________

Example 2

Active Immunization In Mice

Active Immunization with the Recombinant Vaccinia Virus Expressing NS1CTL Epitope is Protective in Mice

In Example 1, results were presented that demonstrated an influenza ANS1 cross-reactive cytotoxic T lymphocyte response in H-2^(d) mice. Theability of an NS1 specific CTL clone to passively protect mice afteradoptive transfer of the NS1 specific CTL clone was reported. There weresignificant decreases in lung virus titers of mice challenged withinfluenza A viruses of all three subtypes i.e., H1, H2 and H3. Theinfluenza A NS1 gene was the kind gift of Dr. Robert Lamb ofNorthwestern University and the gene was truncated and expressed invaccinia virus. The results shown in Table 5 indicate that a CTL epitoperecognized by an influenza A NS1 cross-reactive CTL clone was locatedbetween amino acids 1-40 of NS1.

                  TABLE 5                                                         ______________________________________                                        Recognition of Epitope(s) on Influenza A Non-Structural                       Protein by Virus-Stimulated CTL from BALB/c Virus-Immune                      Spleen Cells in Bulk Culture                                                  Percent specific lysis at E:T                                                 Target Cells Infected With:                                                                   20.0       100    50                                          ______________________________________                                        A/PR/8          71.9       56.9   47.2                                        VAC-entire NS1  74.1       40.3   14.9                                        VAC2 (aal-167)  41.0       25.2   12.3                                        VAC10 (aal-40)  43.5       19.4   16.2                                        Vaccinia alone  -1.6       5.0    4.3                                         Uninfected      13.7       6.5    4.9                                         ______________________________________                                    

The results in Table 5 demonstrate that as a positive controlvirus-stimulated immune spleen cells killed virus-infected target cellsand did not significantly kill uninfected target cells; moreover, theresults demonstrate that target cells infected with recombinant vacciniaviruses expressing various regions of the NS1 gene including the VAC-10recombinant with amino acids 1-40 were significantly killed by theinfluenza virus stimulated PBMC. These results indicate that following asingle infection with influenza A virus, significant memory CTL activityto epitopes on influenza A NS1 is present in H-2^(d) mice.

Active Immunization with VAC/NS1 Recombinant Virus

It was important to determine if it was possible to actively immunizemice with a recombinant virus containing an NS1 gene segment to protectagainst influenza virus. An NS1-specific cross-reactive CD8+CTLresponses had been detected in bulk cultures and at the clonal level,and protection in passive immunization studies with an NS1-specificclone had been shown, but active immunization with a vaccine had notbeen carried out.

Mice were immunized with 0.2 ml (1×10⁸ PFU/ml) of a control vacciniavirus or the recombinant vaccinia-10 which contained the gene segmentencoding influenza A NS1 amino acids 1-40. A second dose of 0.2 ml withthe same amount of virus was administered IP to the two groups of micethree weeks later. Five weeks after the initial dose and two weeksfollowing the second dose spleen cells were removed restimulated invitro with A/PR/8 and then used in a CTL assay.

The results in Table 6 indicate that influenza A virus-specific CTLmemory was induced following administration with the vaccinia-10recombinant and boosting in vitro with influenza A virus. These cellskilled influenza A virus infected target cells, and uninfected targetcells were not lysed. The spleen cells of the control mice that wereprimed with vaccinia virus not containing the NS1 gene, wererestimulated with live influence virus but their cells did notspecifically lyse influenza A virus infected target cells. These resultsindicate that the priming of influenza A virus NS1 specific T cellsresulted in the subsequent generation of influenza A virus cytotoxic Tlymphocytes following repeat stimulation with influenza A virus.

                  TABLE 6                                                         ______________________________________                                        Vaccinia Virus Containing Gene Encoding NS1 aal-40 Induces                    Influenza Virus CTL Memory in BALB/c (H-2.sup.d) Mice                         Lysis of H-2.sup.d Target Cells                                               1°  2°        A/PR/8                                            In Vivo    In Vitro                                                                              E:T      infected                                                                            Uninfected                                  ______________________________________                                        VAC-10     A/PR/8  30       11.2  -1.3                                        (NS1-              10       -1.0  4.4                                         segment)                                                                      Vaccinia   A/PR/8  30       -1.0  1.6                                                            10       1.8   5.6                                         ______________________________________                                    

Seven weeks after the original immunization, mice in these groups werechallenged with 4×10³ plaque forming units of virulent influenza A/PR/34(H1N1) virus administered in 50 μl volumes intranasally. The resultsfrom these experiments demonstrated that mice immunized with VAC/1 weresignificantly protected against lethal challenge with influenza A viruscompared to mice given vaccinia virus alone (P<0.05). More specifically,the survival rate of VAC/NS1 immunized mice observed at 9-16 dayspost-infection was between 70-80%. The survival rate of mice infectedwith vaccinia virus alone (negative control) was about 20% during thesame time period. It was important to determine if active immunizationwould induce protection against disease and death because Stitz et al.reported that a recombinant vaccinia virus which expressed thenucleoprotein (NP) gene of influenza A virus contained a CTL epitope didnot protect when a recombinant vaccinia virus containing the gene forinfluenza nucleoprotein was used to immunize mice, even though a CTLclone specific for NP epitope was protective in passive transferexperiments.

The nucleoprotein gene induced influenza A virus memory CTL's invaccinated mice but did not induce protection against subsequentinfection(s). The results presented herein demonstrate that mice whichhad received the vaccinia expressing a segment of the influenza A NS1gene had CTL memory responses and were protected against lethalchallenge. In conclusion, these results demonstrate that influenza A NS1virus contains protective CTL epitope(s) and can be used as a vaccine toprotect against influenza mortality in mice. The results below indicatethat humans also have influenza A NS1-specific CTL's.

Example 3

Generation & Characterization of a Human

Influenza NS1-Specific T Cell Line

Method of Establishing Human NS1-Specific T Cell Line #77

Human PBMC were stimulated in bulk culture by exposure to influenzaA/PR/8/34 (H1N1) infected autologous stimulator cells (1.6×10⁶) whichhad been infected with virus at a multiplicity of infection of 10 to 1for ninety minutes. After two washes these cells were added to 7×10⁶responder PBMC in AIM V medium containing 15% human AB serum. Followingsix days of culture, the cells were tested for their ability to lyseautologous EBV-transformed B cells in a standard 51 chromium releaseassay. Table 7 demonstrates results at two effector: target ratios, 50and 10:1. There is specific lysis of the autologous target cellsinfected with influenza virus.

                  TABLE 7                                                         ______________________________________                                        Percent Specific Lysis of Target Cells at E:T Ratio                           Effector cells                                                                              Uninfected       A/PR/8 Infected                                ______________________________________                                        A/PR/8 Stimulated                                                                           50     10        50    10                                                     10.7   7.9       45.6  33.9                                     ______________________________________                                    

On day 8, cells from the original culture were re-stimulated withx-irradiated autologous PBMC which had been infected with influenza Avirus, similar to the methods used on day 0. On day 14, alimiting-dilution was performed and 60 wells each received either 10cells, or 30 cells per well in AIM V media, with 10% fetal calf serum10% T cell growth factor 0.1 microgram per ml of anti-CD3 mab andx-irradiated autologous PBMC cells. Fifteen days later, positive growthwas seen in 27 of the 60 wells that were seeded at 10 cells per well,and 52 wells of the 60 that were seeded at 30 cells per well. Threewells were positive in cytotoxicity experiments and the cells in well#77 lysed influenza virus infected autologous BLCL but has very littlelytic activity on the human NK sensitive cell K562 (13.1% vs. 2.1%). Thenext day, the cells in well #77 were fed and expanded by beingre-stimulated every 14 days with the addition of x-irradiated PBMC andanti-CD3 antibody in the presence of AIM V medium and 10% fetal bovineserum and 10% T cell growth factor. The results in Table 8 show thekilling activity of cells from well #77 on autologous BLCL target cellinfected with the vaccinia/1 recombinant virus that contained the NS1gene of influenza A virus infected with influenza A virus A/PR/8/34, butthey did not kill target cells infected with vaccinia virus along orvaccinia viruses expressing the influenza A virus hemagglutinin gene,the nucleo protein gene. The results indicate that cells from well #77specifically killed autologous target cells that expressed the influenzaA NS1 gene.

                  TABLE 8                                                         ______________________________________                                        Lysis of Autologous B-LCL Target Cells Infected with                          Recombinant Vac/Influenza Viruses by Clone/Line #77                           Effector                                                                      Cells                    VAC                 Unin-                            #77   ET    A/PR/8  VAC  HA    VAC NP VAC NS1                                                                              fected                           ______________________________________                                        10      55:0    -4.9   -7.9  -11.4  32.1   -19.5                              ______________________________________                                    

Three weeks later, the T cell line #77 was tested on target cellsinfected with an influenza A (A/PC/1/73, H3 subtype) virus. These targetcells were also killed to a high degree, as were target cells infectedwith the vaccinia recombinant virus expressing the NS1 truncated gene,but #77 cells did not kill target cells infected with recombinantvaccinia viruses expressing the other genes, hemagglutinin ornucleoprotein, of the influenza A virus. These results demonstrate thatclone/line #77 specifically kills autologous cells expressing epitope(s)localized within amino acids 1-167 of the influenza A NS1 protein.

                  TABLE 9                                                         ______________________________________                                        Specific-Lysis by Clone #77 of Autolosous B-LCL Infected                      with Vaccinia, Expressing Truncated Influenza A Virus NS1                     Gene Segments                                                                 Percent specific-lysis of target cells                                        Effector                               Influenza                              Cells       VAC-entire NS1                                                                             VAC-NS1                                                                              VAC-NS1                                                                              A/PC/73                                #77   ET    1-237aa      (aal-167)                                                                            (aal-81)                                                                             (H3N2)                                 ______________________________________                                        10      71.4         67.1     34.4   75.2                                     ______________________________________                                    

HLA Restriction of Influenza A NS1 Specific Cytotoxic T LymphocyteActivity

Experiments were performed using allogeneic target cells which containedpartially matched HLA alleles to define the HLA restriction allele usedby the NS1 specific T cell line #77. The results of the first experimentshown in Table 10 demonstrate that allogeneic target cells that shareHLA A1 and B8 alleles were killed and that target cells that shared B44or CW5 were not killed. In the second experiment partially matched B-LCLtarget cells from three different donors were used, two of which sharedonly A1 and one of which shared only the B8 allele. The resultsdemonstrate that allogenic target cells bearing the HLA B8 allele arekilled, but those that shared only A1 were not. In summary, theseresults clearly demonstrate the existence of a human Class I restricted,influenza A virus NS1 subtype cross-reactive cytotoxic T lymphocyteresponses.

                  TABLE 10                                                        ______________________________________                                        HLA Class I Restriction of #77, NS1-Specific                                  Human CTL Percent Specific Lysis of HLA                                       Partially Matched Cells                                                       HLA Shared          VAC-/NS1 (entire)                                         ______________________________________                                        A.                                                                            Autologous                                                                    (A1        B8      B44 CW5) 69.7                                              A1         B8               64.0                                                                 B44 CW5  -7.1                                              A1         B8               73.3                                              B.                                                                            Autologous                  78.5                                              A1                          -2.1                                                         B8               59.6                                              A1                          -0.7                                              ______________________________________                                    

Equivalents

Those skilled in the art will know, or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the invention described herein. These and otherequivalents are intended to be encompassed by the following claims.

What is claimed is:
 1. A method for immunizing an individual againstdisease caused by infection by influenza A virus comprisingadministering to the individual an effective amount of an influenza Avirus NS1 protein or an influenza A virus NS1 protein derivative inwhich amino acids have been deleted, inserted or substituted withoutessentially detracting from the immunological properties thereof, in acarrier such that said NS1 protein or NS1 protein derivative stimulatesa protective NS1-specific cytotoxic T cell response.
 2. The method ofclaim 1 wherein the NS1 protein derivative consists essentially of anNS1 T cell epitope.
 3. The method of claim 1 wherein the NS1 protein isadministered.